JP3863893B2 - Patch for microarray reaction chamber having adhesive support and two or more adhesive materials - Google Patents

Patch for microarray reaction chamber having adhesive support and two or more adhesive materials Download PDF

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JP3863893B2
JP3863893B2 JP2004363169A JP2004363169A JP3863893B2 JP 3863893 B2 JP3863893 B2 JP 3863893B2 JP 2004363169 A JP2004363169 A JP 2004363169A JP 2004363169 A JP2004363169 A JP 2004363169A JP 3863893 B2 JP3863893 B2 JP 3863893B2
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adhesive
patch
reaction chamber
support
microarray
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JP2005181320A (en
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廷 健 李
憲 周 李
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Samsung Electronics Co Ltd
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    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • B29C66/1122—Single lap to lap joints, i.e. overlap joints
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
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    • B29C66/51—Joining tubular articles, profiled elements or bars; Joining single elements to tubular articles, hollow articles or bars; Joining several hollow-preforms to form hollow or tubular articles
    • B29C66/53—Joining single elements to tubular articles, hollow articles or bars
    • B29C66/534—Joining single elements to open ends of tubular or hollow articles or to the ends of bars
    • B29C66/5346—Joining single elements to open ends of tubular or hollow articles or to the ends of bars said single elements being substantially flat
    • B29C66/53461—Joining single elements to open ends of tubular or hollow articles or to the ends of bars said single elements being substantially flat joining substantially flat covers and/or substantially flat bottoms to open ends of container bodies
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C66/00—General aspects of processes or apparatus for joining preformed parts
    • B29C66/50—General aspects of joining tubular articles; General aspects of joining long products, i.e. bars or profiled elements; General aspects of joining single elements to tubular articles, hollow articles or bars; General aspects of joining several hollow-preforms to form hollow or tubular articles
    • B29C66/51—Joining tubular articles, profiled elements or bars; Joining single elements to tubular articles, hollow articles or bars; Joining several hollow-preforms to form hollow or tubular articles
    • B29C66/54—Joining several hollow-preforms, e.g. half-shells, to form hollow articles, e.g. for making balls, containers; Joining several hollow-preforms, e.g. half-cylinders, to form tubular articles
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/34—Microscope slides, e.g. mounting specimens on microscope slides
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • G01N1/00—Sampling; Preparing specimens for investigation
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Description

本発明はマイクロアレイの反応チャンバ用パッチ及びそれを使用する方法に関する。   The present invention relates to microarray reaction chamber patches and methods of using the same.

一般的に“マイクロアレイ”とは、固体基板上にポリヌクレオチドまたは蛋白質のような重合体のグループが高密度に固定化されているものであり、前記重合体分子はそれぞれ一定の領域に固定化されている。このようなマイクロアレイは当業界で公知のものであり、例えば、特許文献１及び２に開示されている。前記マイクロアレイの例には蛋白質用マイクロアレイやポリヌクレオチドアレイが含まれる。ここに、マイクロアレイ上で反応させるための一定の条件を提供する空間を、“マイクロアレイの反応チャンバ”とすれば、従来のマイクロアレイの反応チャンバは、あらかじめ製作されているものではなく、通常反応を行う前にマイクロアレイ基板とカバーガラスまたはパッチと結合されて形成されていた。   In general, a “microarray” is a group of polymers such as polynucleotides or proteins immobilized on a solid substrate at a high density, and each of the polymer molecules is immobilized in a certain area. ing. Such microarrays are known in the art and are disclosed in, for example, Patent Documents 1 and 2. Examples of the microarray include a protein microarray and a polynucleotide array. Here, if a space that provides a certain condition for the reaction on the microarray is referred to as a “microarray reaction chamber”, the reaction chamber of the conventional microarray is not pre-fabricated and performs a normal reaction. Previously it was formed by combining with a microarray substrate and a cover glass or patch.

図１を参照して、カバーガラスを使用して反応チャンバを形成させる過程の一例を説明すると、次のようになる。まず、プローブポリヌクレオチド２が固定化されているポリヌクレオチドアレイ１上に、標的ポリヌクレオチドを含む試料３を、マイクロピペット４を利用して適当量滴下し、滴下された試料滴３上にカバーガラス５を覆う。それにより、カバーガラスとマイクロアレイの基板との間の空間が反応チャンバの機能を行い、ハイブリダイゼーションを実行させることができる。
米国特許第５，４４５，９３４号明細書 米国特許第５，７４４，３０５号明細書 With reference to FIG. 1, an example of a process of forming a reaction chamber using a cover glass will be described as follows. First, an appropriate amount of a sample 3 containing the target polynucleotide is dropped on the polynucleotide array 1 on which the probe polynucleotide 2 is immobilized using a micropipette 4, and a cover glass is placed on the dropped sample drop 3. 5 is covered. Thereby, the space between the cover glass and the substrate of the microarray performs the function of the reaction chamber, and hybridization can be executed.
US Pat. No. 5,445,934 US Pat. No. 5,744,305

この方法は、カバーガラスを利用するためにコストダウンできるという長所がある。しかし、標的ポリヌクレオチドを含む試料の投入量を調節し難く、温度が高くて相対湿度が飽和されていない状態では試料が蒸発する場合があり、反応結果として得られる光学的信号を含む信号にも影響を及ぼして再現性のある結果を得難い短所がある。   This method has an advantage that the cost can be reduced because the cover glass is used. However, it is difficult to adjust the input amount of the sample containing the target polynucleotide, and the sample may evaporate in a state where the temperature is high and the relative humidity is not saturated, and the signal including the optical signal obtained as a reaction result is also included. There is a disadvantage that it is difficult to obtain reproducible results.

また、従来のパッチを利用して反応チャンバを形成する方法もある。一般的にこのようなパッチは、基板と接着される接着部と、前記接着部が付着されている支持部とで構成されており、前記基板と接着された場合に反応チャンバを形成する空間であるチャンバ形成部が設けられている。これを、図２を用いて説明する。図２Ａは、パッチの側面断面図であり、試料注入口付きの支持部１０と、それに付着されている接着部９とで構成され、これによって反応チャンバ形成部２２が確保される。図２Ｂは、このようなパッチを基板上に付着して形成された反応チャンバに反応液を添加する場合の状態を示す平面透視図である。このような反応チャンバは、取っ手８を利用して前記パッチを基板上に付着することによって形成される。このように形成された反応チャンバで反応を行うには、試料注入口７にマイクロピペット４を利用して試料を注入するが、従来の反応チャンバでは、前記チャンバ形成部の形態が最適化されずにバブル６が発生する短所がある。また、前記パッチは１種の接着物質のみ使用するため、基板と強く接着する物質を使用することによって漏れ現象を防止できるが、反応が完了した後で脱着する場合に脱着し難く、脱着したとしても接着物質が残留する場合があった。また、支持部１０との接着力が弱くて接着部９と支持部１０とが分離される場合がある。さらに、チャンバの容量は、前記接着部の厚さをさらに厚くすることで調節できるが、１種の接着物質のみ使用するため、さらに厚くする場合に厚さを一定にすることが困難であった。   There is also a method of forming a reaction chamber using a conventional patch. In general, such a patch is composed of an adhesive portion bonded to a substrate and a support portion to which the adhesive portion is attached, and is a space that forms a reaction chamber when bonded to the substrate. A chamber forming part is provided. This will be described with reference to FIG. FIG. 2A is a side cross-sectional view of the patch, which includes a support portion 10 with a sample injection port and an adhesive portion 9 attached to the support portion 10, thereby securing a reaction chamber forming portion 22. FIG. 2B is a plan perspective view showing a state in which a reaction solution is added to a reaction chamber formed by attaching such a patch on a substrate. Such a reaction chamber is formed by depositing the patch on the substrate using the handle 8. In order to perform the reaction in the reaction chamber thus formed, a sample is injected into the sample inlet 7 using the micropipette 4. However, in the conventional reaction chamber, the form of the chamber forming portion is not optimized. However, there is a disadvantage that the bubble 6 is generated. In addition, since the patch uses only one kind of adhesive substance, it is possible to prevent the leakage phenomenon by using a substance that adheres strongly to the substrate. In some cases, the adhesive material remained. Moreover, the adhesive force with the support part 10 is weak, and the adhesive part 9 and the support part 10 may be separated. Further, the volume of the chamber can be adjusted by further increasing the thickness of the bonding portion, but since only one kind of adhesive material is used, it is difficult to make the thickness constant when the thickness is further increased. .

したがって、本発明の目的は、基板から容易に脱着されつつも支持部と接着部とが分離されず、かつ接着部の厚さを安定的に調節できるマイクロアレイ反応チャンバ用パッチを提供することである。   Accordingly, an object of the present invention is to provide a patch for a microarray reaction chamber that can be easily detached from a substrate but the support part and the adhesive part are not separated and the thickness of the adhesive part can be adjusted stably. .

また、本発明の目的は、前記マイクロアレイパッチの使用方法を提供することである。   Another object of the present invention is to provide a method for using the microarray patch.

本発明者らは前記のような従来技術に基づいて、基板から容易に脱着できるが支持部と接着部とが分離されず、かつ接着部の厚さを安定して調節できるマイクロアレイ反応チャンバ用パッチについて研究し、接着部に接着部支持体を導入して２つ以上の接着物質を使用することで、前記問題を解決できることを見出し、本発明を完成するに至った。   The inventors of the present invention have a microarray reaction chamber patch that can be easily detached from the substrate based on the prior art as described above, but the support part and the adhesive part are not separated and the thickness of the adhesive part can be stably adjusted. The present invention was completed by finding that the above problem can be solved by introducing an adhesive support to the adhesive part and using two or more adhesive substances.

本発明のマイクロアレイの反応チャンバ用パッチ使用する場合、試料の漏れを防止でき、かつ脱着時には支持部を離脱させずに基板から容易に脱着させうる。また、反応チャンバの容量を容易に調整できる。   When the patch for the reaction chamber of the microarray of the present invention is used, the sample can be prevented from leaking, and can be easily detached from the substrate without detaching the support portion during detachment. In addition, the capacity of the reaction chamber can be easily adjusted.

本発明の第一は、試料注入口付きの支持部と、前記支持部に接着し、かつマイクロアレイ基板に付着しうる接着力を有する接着部とからなり、前記支持部及び接着部によって限定された反応チャンバ形成部を有するマイクロアレイの反応チャンバ用パッチにおいて、
前記接着部は、接着物質を両面に付着した接着部支持体が積層されて前記支持部に付着されており、前記接着部支持体と接着部支持体との間および前記接着部支持体と前記支持部との間を付着させる第１接着物質の接着力が、前記接着部支持体とマイクロアレイ基板との間を付着させる第２接着物質の接着力に比べて３３ｏｚ／ｉｎ（ＡＳＴＭ Ｄ３３３０方法）以上であることを特徴とする反応チャンバ用パッチである。 The first aspect of the present invention includes a support portion with a sample injection port and an adhesive portion that has an adhesive force that adheres to the support portion and can adhere to the microarray substrate, and is limited by the support portion and the adhesive portion. In a microarray reaction chamber patch having a reaction chamber forming portion,
The adhesive part is formed by laminating an adhesive part support body having an adhesive substance attached to both surfaces thereof and being attached to the support part, and between the adhesive part support body and the adhesive part support body and the adhesive part support body and the The adhesive force of the first adhesive substance that adheres to the support part is 33 oz / in (ASTM D3330 method) or more compared to the adhesive force of the second adhesive substance that adheres between the adhesive part support and the microarray substrate. This is a reaction chamber patch.

本発明において、前記支持部は、接触角８０゜以上の疎水性物質で構成されることが好ましい。このような前記疎水性物質としては、疎水性プラスチックまたは疎水性処理されたシリコンがある。前記疎水性物質としては、ポリエチレン、ポリプロピレン、ポリカーボネート、ポリスチレン、ポリメチルメタクリレート、ポリジメチルシロキサン、ポリ塩化ビフェニル、疎水性に処理されたシリコンウェーハ、疎水性に処理されたＩＴＯガラス、またはそれらの組合わせなどがあるが、これらに限定されるものではない。   In the present invention, the support part is preferably made of a hydrophobic material having a contact angle of 80 ° or more. Such hydrophobic materials include hydrophobic plastics or hydrophobically treated silicon. Examples of the hydrophobic material include polyethylene, polypropylene, polycarbonate, polystyrene, polymethyl methacrylate, polydimethylsiloxane, polychlorinated biphenyl, a hydrophobically treated silicon wafer, a hydrophobically treated ITO glass, or a combination thereof. However, it is not limited to these.

本発明において、前記接着部支持体は、ポリエチレンテレフタレート、ポリエチレン、及びポリエステルからなる群から選択される。しかし、これら例に限定されるものではない。また、前記接着部支持体は、本発明のパッチが使われる目的によって一つまたは複数を使用してもよく、その材質もそれぞれ同じでも異なってもよい。   In the present invention, the adhesive support is selected from the group consisting of polyethylene terephthalate, polyethylene, and polyester. However, it is not limited to these examples. One or a plurality of the adhesive support members may be used depending on the purpose for which the patch of the present invention is used, and the materials may be the same or different.

本発明において、第１接着物質とは、前記接着部支持体と接着部支持体との間、または前記接着部支持体と前記支持部との間を付着させる接着物質をいう。前記第１接着物質は、第２接着物質に比べて３３ｏｚ／ｉｎ以上の接着力を持つものであることが好ましい。また、前記第１接着物質は、接着力が４５ｏｚ／ｉｎ以上である。   In the present invention, the first adhesive substance refers to an adhesive substance that adheres between the adhesive part support and the adhesive part support or between the adhesive part support and the support part. The first adhesive material preferably has an adhesive force of 33 oz / in or more as compared with the second adhesive material. The first adhesive substance has an adhesive strength of 45 oz / in or more.

本発明において、第２接着物質とは、本発明のパッチとマイクロアレイ基板との間を付着させる接着物質である。前記第２接着物質の接着力は、望ましくは、前記第２接着物質は再接着性を持つものであって、接着力が１２ｏｚ／ｉｎないし２５ｏｚ／ｉｎである。   In the present invention, the second adhesive substance is an adhesive substance that adheres between the patch of the present invention and the microarray substrate. The adhesive strength of the second adhesive material is preferably such that the second adhesive material has re-adhesiveness, and the adhesive strength is 12 oz / in to 25 oz / in.

本発明の反応チャンバ用パッチは、試料注入口付きの支持部と、前記支持部に接着し、かつマイクロアレイ基板に付着しうる接着力を有する接着部とからなるが、前記支持部と接着部とによって形成される反応チャンバ形成部は、その形状に限定はないが、好ましくは流線型である。   The reaction chamber patch of the present invention comprises a support portion with a sample inlet and an adhesive portion that adheres to the support portion and has an adhesive force that can adhere to the microarray substrate. The shape of the reaction chamber forming part formed by is not limited, but is preferably streamlined.

本発明において、「マイクロアレイ」とは、基板上にポリヌクレオチドまたは蛋白質などの生体分子が高密度に固定化されているものであって、前記生体分子はそれぞれ一定の領域に固定化されている。マイクロアレイとしては、例えば、前記の特許文献１や２に開示されているが、本発明はこれらに限定されず、従来公知のマイクロアレイに応用できる。なお、従って、本発明の反応チャンバ用パッチは、蛋白質用マイクロアレイやポリヌクレオチドアレイにも応用できる。   In the present invention, a “microarray” is a substrate in which biomolecules such as polynucleotides or proteins are immobilized at a high density, and each of the biomolecules is immobilized in a certain region. Examples of the microarray are disclosed in Patent Documents 1 and 2 described above, but the present invention is not limited to these and can be applied to conventionally known microarrays. Therefore, the reaction chamber patch of the present invention can also be applied to protein microarrays and polynucleotide arrays.

前記マイクロアレイがポリヌクレオチドマイクロアレイである場合を例にとり、本発明のパッチの使用方法を説明する。   Taking the case where the microarray is a polynucleotide microarray as an example, a method of using the patch of the present invention will be described.

まず、本発明のパッチを、プローブポリヌクレオチドが固定化されているポリヌクレオチドアレイ基板上に付着すると、該基板上に反応チャンバが形成される。前記反応チャンバに、試料注入口から標的試料を含むハイブリダイゼーション液を注入し、一定の条件下でハイブリダイゼーション行う。ハイブリダイゼーションが終わってから洗浄液で洗浄し、十分に乾燥し、ハイブリダイゼーションの程度をスキャナによりスキャニングすれば、その結果を測定することができる。   First, when the patch of the present invention is attached to a polynucleotide array substrate on which a probe polynucleotide is immobilized, a reaction chamber is formed on the substrate. A hybridization solution containing a target sample is injected into the reaction chamber from a sample injection port, and hybridization is performed under certain conditions. After the hybridization is completed, the result can be measured by washing with a washing solution, sufficiently drying, and scanning the degree of hybridization with a scanner.

以下、本発明の反応チャンバ用パッチの一例を図面を参照してさらに具体的に説明する。図３は、本発明のマイクロアレイ反応チャンバ用パッチの側面図である。図３に図示すように、本発明の反応チャンバ用パッチは、支持部１０とそれに付着されている接着部９とで構成されるが、前記接着部９は、第１接着物質１１が接着部支持体１２の上下面に付着されている第１接着層１３と、上面には第１接着物質１１が付着されており、下面には第２接着物質１４が付着されている第２接着層１５とで構成されている。前記接着部９は、前記第１接着層１３の上面に付着されている第１接着物質１１によって前記支持部に付着され、前記第２接着層１５の下面に付着されている第２接着物質１４によってマイクロアレイ基板上に付着することができる。また、本発明のパッチには、マイクロアレイ基板上に付着させた時に反応チャンバを形成できる反応チャンバ形成部２２が備えられている。このような反応チャンバ形成部２２は、前記接着部を一定に切り取ることによって形成される。前記接着部の厚さは、使用される前記接着部支持体の個数によって適宜選択することができる。したがって、前記接着部支持体の個数を調整することによって前記接着部の厚さを調整でき、それにより反応チャンバ形成部の容量を調整できる。なお、更に第ｎ番目の第ｎ接着層まで積層する場合には、第１接着物質の接着力が、前記接着部支持体とマイクロアレイ基板との間を付着させる第ｎ接着物質の接着力に比べて３３ｏｚ／ｉｎ（ＡＳＴＭ Ｄ３３３０方法）以上とすることが好ましく、また、第ｎ接着物質の接着力は１２ｏｚ／ｉｎないし２５ｏｚ／ｉｎであることが好ましい。この場合、第ｎ−１接着層までは、第一接着層と同じものを使用することができる。   Hereinafter, an example of the reaction chamber patch of the present invention will be described more specifically with reference to the drawings. FIG. 3 is a side view of the patch for the microarray reaction chamber of the present invention. As shown in FIG. 3, the reaction chamber patch according to the present invention includes a support portion 10 and an adhesive portion 9 attached to the support portion 10. The adhesive portion 9 includes a first adhesive substance 11 and an adhesive portion. A first adhesive layer 13 attached to the upper and lower surfaces of the support 12, a first adhesive material 11 attached to the upper surface, and a second adhesive layer 15 attached to the second adhesive material 14 on the lower surface. It consists of and. The adhesive portion 9 is attached to the support portion by the first adhesive material 11 attached to the upper surface of the first adhesive layer 13 and the second adhesive material 14 attached to the lower surface of the second adhesive layer 15. Can be deposited on the microarray substrate. In addition, the patch of the present invention is provided with a reaction chamber forming portion 22 that can form a reaction chamber when attached to the microarray substrate. Such a reaction chamber forming part 22 is formed by cutting out the adhesive part to a certain extent. The thickness of the adhesive part can be appropriately selected depending on the number of the adhesive part supports used. Therefore, the thickness of the bonding part can be adjusted by adjusting the number of the bonding part supports, and thereby the capacity of the reaction chamber forming part can be adjusted. In the case of further stacking up to the nth nth adhesive layer, the adhesive force of the first adhesive substance is compared with the adhesive force of the nth adhesive substance that adheres between the adhesive support and the microarray substrate. It is preferable to set to 33 oz / in (ASTM D3330 method) or more, and the adhesive strength of the nth adhesive substance is preferably 12 oz / in to 25 oz / in. In this case, the same material as the first adhesive layer can be used up to the (n-1) th adhesive layer.

本発明の反応チャンバ用パッチの製造方法に限定はないが、例えば、次のような過程によって製造できる。まず、図４に示すように、接着部支持体１２の上面及び下面に第１接着物質１１を付着させて第１接着層１３を形成し、他の接着部支持体１２の上面には第１接着物質１１を付着させ、下面には第２接着物質１４を付着させて第２接着層１５を形成する。次いで、それら第１接着層１３と第２接着層１５とを第１接着物質１１間の接着力を利用して接合させ、接着部を形成する。次に、図５に示すように、適当な大きさ及び形態で前記接着部の一部１７を切り取って反応チャンバ形成部２２を形成する。前記反応チャンバ形成部２２が設けられた前記接着部を試料注入口付きの支持部に接着すると、本発明のパッチが形成される。   Although there is no limitation in the manufacturing method of the patch for reaction chambers of this invention, it can manufacture by the following processes, for example. First, as shown in FIG. 4, a first adhesive layer 11 is formed by attaching a first adhesive material 11 to the upper surface and the lower surface of the bonding portion support 12, and the first bonding layer 13 is formed on the upper surface of the other bonding portion support 12. An adhesive substance 11 is attached, and a second adhesive layer 14 is formed on the lower surface to form a second adhesive layer 15. Next, the first adhesive layer 13 and the second adhesive layer 15 are joined using the adhesive force between the first adhesive substances 11 to form an adhesive portion. Next, as shown in FIG. 5, the reaction chamber forming portion 22 is formed by cutting out a part 17 of the bonding portion with an appropriate size and shape. When the bonding portion provided with the reaction chamber forming portion 22 is bonded to a support portion with a sample inlet, the patch of the present invention is formed.

図６及び図７は、本発明の反応チャンバ用パッチの使用方法の一例を示す図面である。図６ａは、本発明の反応チャンバ用パッチがポリヌクレオチドマイクロアレイ基板に付着された状態を示す平面図の透視図である。本発明の反応チャンバ用パッチを前記マイクロアレイ基板上に付着させることによって、反応チャンバが形成される。これに、試料注入口７を通じて反応液を注入すれば、矢印で表したように試料が前記反応チャンバに分配される。図６ｂは、図６ａの部分側面図である。   6 and 7 are diagrams showing an example of a method of using the reaction chamber patch of the present invention. FIG. 6a is a perspective view of a plan view showing the reaction chamber patch of the present invention attached to a polynucleotide microarray substrate. The reaction chamber is formed by attaching the reaction chamber patch of the present invention on the microarray substrate. When a reaction solution is injected into the sample through the sample injection port 7, the sample is distributed to the reaction chamber as indicated by an arrow. 6b is a partial side view of FIG. 6a.

次いで、試料注入口７をテープ２０を利用して密閉し、適当なハイブリダイゼーション条件、例えば、３７℃で１６時間培養する（図７ａ）。図７ｂは、本発明のパッチが基板に付着されて形成された反応チャンバ中の試料を注入した場合を示す反応チャンバの側面の部分断面図である。前記反応チャンバは、前記パッチの接着部と基板とが前記第２接着物質によって接合されることで形成される。基板上に固定化されているプローブポリヌクレオチド１８は、試料中の標的ポリヌクレオチド１９とハイブリダイズする。ハイブリダイゼーションの間、前記パッチから試料が漏れてはならないが、前記第２接着物質は、除去時に容易に脱着され、かつ基板上に残留物質を残してはならない。このような条件を満たすには、前記第２接着物質は、鋼鉄に対する接着力を基準にして、１２〜２５ｏｚ／ｉｎ（ＡＳＴＭ Ｄ３３３０方法）の接着力を持つことが望ましい。また、前記第１接着物質は、前記支持部と強く接着させて前記パッチを脱着させる際に、第２接着物質より先に離脱されてはならない。前記第１接着物質は鋼鉄に対する接着力を基準に４５ｏｚ／ｉｎ（ＡＳＴＭ Ｄ３３３０方法）の接着力を持つことが望ましい。   Next, the sample inlet 7 is sealed using the tape 20, and cultured for 16 hours at an appropriate hybridization condition, for example, 37 ° C. (FIG. 7a). FIG. 7 b is a partial cross-sectional view of the side of the reaction chamber showing a case where a sample in the reaction chamber formed by attaching the patch of the present invention to the substrate is injected. The reaction chamber is formed by bonding an adhesive portion of the patch and a substrate with the second adhesive material. The probe polynucleotide 18 immobilized on the substrate hybridizes with the target polynucleotide 19 in the sample. During hybridization, the sample should not leak from the patch, but the second adhesive material should be easily desorbed upon removal and leave no residual material on the substrate. In order to satisfy such a condition, the second adhesive material preferably has an adhesive force of 12 to 25 oz / in (ASTM D3330 method) based on the adhesive force to steel. The first adhesive material should not be detached prior to the second adhesive material when the patch is detached and attached to the support portion. The first adhesive material preferably has an adhesive strength of 45 oz / in (ASTM D3330 method) based on the adhesive strength to steel.

以下本発明を実施例を通じてさらに詳細に説明する。しかし、それら実施例は本発明を例示的に説明するためのものであって、本発明の範囲がそれら実施例に限定されるものではない。   Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

次に実施例を挙げて本発明を具体的に説明するが、これらの実施例は何ら本発明を制限するものではない。   EXAMPLES Next, although an Example is given and this invention is demonstrated concretely, these Examples do not restrict | limit this invention at all.

（実施例１）
第１接着物質及び第２接着物質の接着力がパッチ特性に及ぶ影響
本実施例では、接着力が１００ｏｚ／ｉｎである第１接着物質が上面及び下面に付着された厚さ１２０μｍのテープ（３Ｍ社製、商品名「９４９５ＭＰＴＭテープ」）を第１接着層として使用し、上面は４５ｏｚ／ｉｎの接着力を持つ第１接着物質でコーティングされており、下面は１２ｏｚ／ｉｎの接着力を持つ第２接着物質でコーティングされているテープ（３Ｍ社製、商品名「９４２５ＰＣＴＭ テープ」）を第２接着層として使用した。前記２層の接着層を結合させた後、厚さ２００μｍで試料注入口付きのポリカーボネート材質の支持部に接合させてマイクロアレイ反応チャンバ用パッチを形成した。 Example 1
Effect of Adhesive Force of First Adhesive Material and Second Adhesive Material on Patch Properties In this example, a 120 μm-thick tape (3M) having a first adhesive material having an adhesive force of 100 oz / in attached to the upper and lower surfaces The product name "9495MP TM tape" is used as the first adhesive layer, and the upper surface is coated with a first adhesive material having an adhesive force of 45 oz / in and the lower surface has an adhesive force of 12 oz / in. A tape coated with the second adhesive substance (trade name “9425PC TM tape” manufactured by 3M Co., Ltd.) was used as the second adhesive layer. After the two adhesive layers were bonded, the microarray reaction chamber patch was formed by bonding to a polycarbonate support having a thickness of 200 μm and a sample inlet.

次に、前記パッチをマイクロアレイ基板上に付着して反応チャンバを形成し、標的ポリヌクレオチドが含まれている試料を試料注入口を通じて注入し、３７℃で１６時間ハイブリダイゼーションを行った後、前記パッチを脱着した。前記ハイブリダイゼーション中の試料の漏れ率、脱着満足率及び支持部付着率を調べて記録した。３人の測定者がそれぞれ２０回反復して測定した。試料の漏れ率の基準は、漏れによる試料損失があるかどうかをスキャナ（ＧｅｎｅＰｉｘ ４０００ＢＴＭ，Ａｘｏｎ Ｉｎｓｔｒｕｍｅｎｔｓ，Ｉｎｃ．）で測定し、脱着満足率は、脱着時それぞれの測定者がハイブリダイゼーション後に前記パッチを脱着した時の満足度を平均したものである。満足度の基準は、脱着が不可能なものは０とし、その他の脱着率は、脱着時の接着物質の残留有無及び脱着時パッチの変形なしに脱着されるかどうかによって表示した。すなわち、パッチの残留及び変形なしに容易に脱着されるものを１００と表示した。支持部付着率は、ハイブリダイゼーション後に脱着時、第１接着物質が支持部から全く脱着されないものを１００として測定した。その結果を表１に表す。 Next, the patch is attached onto the microarray substrate to form a reaction chamber, a sample containing the target polynucleotide is injected through a sample inlet, and hybridization is performed at 37 ° C. for 16 hours, and then the patch Was desorbed. The leakage rate, the desorption satisfaction rate and the support adhesion rate of the sample during the hybridization were examined and recorded. Three measurers each measured 20 times. The standard for the leakage rate of the sample is to measure whether there is a sample loss due to leakage with a scanner (GenePix 4000B ™ , Axon Instruments, Inc.). It is the average of satisfaction when desorbing. The degree of satisfaction was set to 0 for those incapable of desorption, and the other desorption rates were indicated by whether or not the adhesive material remained at the time of desorption and whether or not the desorption patch was deformed without deformation. That is, 100 is shown as what can be easily detached without residual or deformation of the patch. The support portion adhesion rate was measured by taking 100 as the value at which the first adhesive substance was not detached from the support portion at the time of desorption after hybridization. The results are shown in Table 1.

Figure 0003863893
Figure 0003863893

表１に表した値は、それぞれ２０回実験の結果を平均したものである。表１に表したように、試料漏れは、接着力が１２ｏｚ／ｉｎ以上の場合には発生しなかった。脱着満足率は、１２ｏｚ／ｉｎないし２３ｏｚ／ｉｎで最適の結果を得た。支持部との付着率は、４５ｏｚ／ｉｎ以上で満足すべき結果を得た。すなわち、試料の漏れもなく脱着満足率もよい第２接着物質の接着力は、１２ｏｚ／ｉｎ〜２３ｏｚ／ｉｎであった。支持部との付着率は、第１接着物質が４５ｏｚ／ｉｎ以上の接着力を持つ場合、前記支持体からあまり脱着されなかった。但し、前記第２接着物質及び第１接着物質の接着力の差は３３ｏｚ／ｉｎ以上でなければならない。このような条件を満たして初めて、前記接着部のマイクロアレイ基板との接着力と支持部との接着力との間に十分な差が出て満足すべき脱着満足率を得られる。   The values shown in Table 1 are obtained by averaging the results of 20 experiments. As shown in Table 1, sample leakage did not occur when the adhesive strength was 12 oz / in or more. The desorption satisfaction rate was 12 oz / in to 23 oz / in, and optimum results were obtained. Satisfactory results were obtained when the adhesion rate with the support was 45 oz / in or more. That is, the adhesive strength of the second adhesive material having a good sample removal rate and a satisfactory desorption rate was 12 oz / in to 23 oz / in. When the first adhesive substance had an adhesive force of 45 oz / in or more, the adhesion rate with the support part was not so much detached from the support. However, the difference in adhesive force between the second adhesive material and the first adhesive material must be 33 oz / in or more. Only when these conditions are satisfied, a satisfactory difference can be obtained between the adhesive force between the adhesive portion and the microarray substrate and the adhesive force between the support portion and a satisfactory desorption satisfaction rate.

（実施例２）
実施例１の結果に基づいて接着力１２ｏｚ／ｉｎの物質がフィルムの一面に、４５ｏｚ／ｉｎの物質がフィルムの他面にそれぞれ接着されて製作されたテープ（３Ｍ社製、商品名「９４２５ＰＣＴＭ テープ」）を第２接着層として使用し、厚さを調整するためにフィルムの両面に１００ｏｚ／ｉｎの接着物質が接着されて製作されたテープ（３Ｍ社製、商品名「９４９５ＭＰＴＭ テープ」）を第１接着層として使用した。９４２５ＰＣＴＭ １枚、９４９５ＭＰＴＭ ２枚を組合わせて厚さ４００μｍの接合されたテープを得て、図８に示すように、支持部として２００μｍのポリカーボネートフィルムと接着してパッチを製作した。図９ａは、得られた接着部から１４ｍｍ×１４ｍｍ大きさの空間を切り取ってチャンバ形成部を形成した前記接着部を表したものであり、図９ｂは、試料を注入するための試料注入口７付きのポリカーボネ―ト支持部を表したものである。図９ａは、チャンバ形成部を上から見下ろしたものであって、従来のチャンバで使われた角部分を除去して流線型に設計した。 (Example 2)
Based on the result of Example 1, a tape manufactured by bonding a substance having an adhesive strength of 12 oz / in to one side of the film and a substance having a 45 oz / in to the other side of the film (trade name “9425PC ™ , manufactured by 3M Co., Ltd.) Tape ") was used as the second adhesive layer, and 100 oz / in adhesive material was bonded to both sides of the film to adjust the thickness (made by 3M, trade name" 9495MP TM tape ") Was used as the first adhesive layer. 9425PC TM 1 sheet, to obtain a joined tape having a thickness of 400μm by combining two 9495MP TM, as shown in FIG. 8 was fabricated patch adhered to the polycarbonate film of 200μm as a support. FIG. 9a shows the adhesion part obtained by cutting a space of 14 mm × 14 mm from the obtained adhesion part to form a chamber formation part, and FIG. 9b shows a sample injection port 7 for injecting a sample. It represents a polycarbonate support with a mark. FIG. 9A is a view of the chamber forming portion as viewed from above, and is designed to be streamlined by removing corner portions used in the conventional chamber.

このように製作されたマイクロアレイのハイブリダイゼーションチャンバに対し試料漏れ率、脱着満足率及び支持部付着率を測定した。その結果を表２に表した。   The sample leakage rate, the desorption satisfaction rate, and the support adhesion rate were measured for the hybridization chamber of the microarray manufactured as described above. The results are shown in Table 2.

Figure 0003863893
Figure 0003863893

表２に表したように、試料漏れ率０％、脱着満足率１００％及び支持部付着率１００％の満足すべき結果を得た。   As shown in Table 2, satisfactory results were obtained with a sample leakage rate of 0%, a desorption satisfaction rate of 100%, and a support portion adhesion rate of 100%.

前記マイクロアレイ反応チャンバを使用して、ポリヌクレオチドアレイにテストを行った。実験条件は次の通りである。   The microarray reaction chamber was used to test polynucleotide arrays. The experimental conditions are as follows.

１）プローブポリヌクレオチド：配列番号１
標的ポリヌクレオチド：配列番号２，５００ｐＭ
スポットピッチ：３００μｍ、スポット直径：１６０μｍ、スポット数：５２８個
２）ハイブリダイゼーション時間及び温度：１６時間、３２℃
３）洗浄時間：Ｉ段階−６Ｘ ＳＳＣで５分、ＩＩ段階−３Ｘ ＳＳＣで５分
４）ＰＭＴ ５７３、５３２ｎｍ波長でグリーンモードで測定（Ｓｃａｎｎｅｒ ＧｅｎｅＰｉｘ ４０００ＢＴＭ，Ａｘｏｎ Ｉｎｓｔｒｕｍｅｎｔｓ，Ｉｎｃ．）
スキャニングの結果、平均蛍光強度９６５５．３１を得た。 1) Probe polynucleotide: SEQ ID NO: 1
Target polynucleotide: SEQ ID NO: 2,500 pM
Spot pitch: 300 μm, spot diameter: 160 μm, number of spots: 528 2) Hybridization time and temperature: 16 hours, 32 ° C.
3) Washing time: 5 minutes for I-stage-6X SSC, 5 minutes for II-stage-3X SSC 4) PMT 573, measured in green mode at 532 nm wavelength (Scanner GenePix 4000B ™ , Axon Instruments, Inc.)
As a result of scanning, an average fluorescence intensity of 9655.31 was obtained.

この実験の結果、本発明のパッチが、ポリヌクレオチドアレイの反応チャンバとしての機能を優秀に行えるということが示された。   As a result of this experiment, it was shown that the patch of the present invention can perform an excellent function as a reaction chamber of the polynucleotide array.

本発明のマイクロアレイの反応チャンバ用パッチは、マイクロアレイを利用した分析方法において、プローブと標的物質とを反応させる段階で反応チャンバを形成させるのに好適に使われ、有用である。   The reaction chamber patch of the microarray of the present invention is suitably used for forming a reaction chamber at the stage of reacting a probe and a target substance in an analysis method using the microarray.

従来のカバーガラスを利用してチャンバを形成する過程の一例を示す図面である。6 is a diagram illustrating an example of a process of forming a chamber using a conventional cover glass. 図２Ａは、従来のチャンバ形成用パッチの側面断面図であり、図２Ｂは、従来のパッチを利用してチャンバを形成する過程の一例を示す図面である。2A is a side cross-sectional view of a conventional chamber forming patch, and FIG. 2B is a diagram illustrating an example of a process of forming a chamber using a conventional patch. 本発明のマイクロアレイ反応チャンバ用パッチの側面図である。It is a side view of the patch for microarray reaction chambers of this invention. 本発明のパッチの接着部を形成する過程を示す図面である。3 is a diagram illustrating a process of forming an adhesive portion of a patch according to the present invention. 本発明の接着部より反応チャンバ形成部を製作する過程を示す図面である。3 is a diagram illustrating a process of manufacturing a reaction chamber forming unit from an adhesive unit according to the present invention. 図６ａは、本発明の反応チャンバ用パッチがポリヌクレオチドマイクロアレイ基板に付着された状態を示す平面図の透視図であり、図６ｂは、図６ａの部分側面図である。6a is a perspective view of a plan view showing a state in which the reaction chamber patch of the present invention is attached to a polynucleotide microarray substrate, and FIG. 6b is a partial side view of FIG. 6a. 本発明のパッチの使用方法の一例を示す図面であり、図７ａは、試料注入口７をテープ２０を利用して密閉したところ、図７ｂは本発明のパッチが基板に付着されて形成された反応チャンバ中の試料を注入した場合を示す反応チャンバの側面の部分断面図である。FIG. 7A is a view showing an example of a method of using the patch of the present invention. FIG. 7A shows a case where the sample inlet 7 is sealed using a tape 20, and FIG. 7B is formed by attaching the patch of the present invention to a substrate. It is a fragmentary sectional view of the side surface of the reaction chamber showing a case where a sample in the reaction chamber is injected. 本発明の実施例２で使われたパッチの接着部を示す図面である。It is drawing which shows the adhesion part of the patch used in Example 2 of this invention. 本発明の実施例２の接着部よりチャンバ形成部を製作する過程を示す図面である。It is drawing which shows the process in which a chamber formation part is manufactured from the adhesion part of Example 2 of this invention.

符号の説明Explanation of symbols

４・・・マイクロピペット、
７・・・試料注入口、
９・・・接着部、
１０・・・支持部、
１１・・・第１接着物質、
１２・・・接着部支持体、
１３・・・第１接着層、
１４・・・２接着物質、
１７・・・切り取り部、
１５・・・第２接着層、
１８・・・プローブポリヌクレオチド、
１９・・・標的ポリヌクレオチド、
２０・・・テープ、
２２・・・反応チャンバ形成部。 4 ... Micropipette,
7 ... Sample inlet,
9: Adhesive part,
10 ... support part,
11 ... first adhesive substance,
12: Adhesive support,
13: first adhesive layer,
14 ... 2 adhesive substance,
17 ... cutting part,
15 ... second adhesive layer,
18 ... probe polynucleotide,
19 ... Target polynucleotide,
20 ... tape,
22 ... Reaction chamber forming part.

Claims (10)

試料注入口付きの支持部と、前記支持部に接着し、かつマイクロアレイ基板に付着しうる接着力を有する接着部とからなり、前記支持部及び接着部によって限定された反応チャンバ形成部を有するマイクロアレイの反応チャンバ用パッチにおいて、
前記接着部は、接着物質を両面に付着した接着部支持体が積層されて前記支持部に付着されており、前記接着部支持体と接着部支持体との間および前記接着部支持体と前記支持部との間を付着させる第１接着物質の接着力が、前記接着部支持体とマイクロアレイ基板との間を付着させる第２接着物質の接着力に比べて３６０Ｎ／ｍ（ＡＳＴＭ Ｄ３３３０方法）以上であることを特徴とする反応チャンバ用パッチ。 A microarray having a reaction chamber forming portion defined by a support portion with a sample injection port and an adhesive portion having an adhesive force that adheres to the support portion and adheres to the microarray substrate. In the reaction chamber patch,
The adhesive part is formed by laminating an adhesive part support body having an adhesive substance attached to both surfaces thereof and being attached to the support part, and between the adhesive part support body and the adhesive part support body and the adhesive part support body and the The adhesive force of the first adhesive substance that adheres to the support part is 360 N / m (ASTM D3330 method) or more compared to the adhesive force of the second adhesive substance that adheres between the adhesive part support and the microarray substrate. A patch for a reaction chamber. 前記支持部は、水接触角８０゜以上の疎水性物質で構成されることを特徴とする請求項１に記載のパッチ。 The patch according to claim 1, wherein the support part is made of a hydrophobic material having a water contact angle of 80 ° or more. 前記疎水性物質は疎水性プラスチックまたは疎水性処理されたシリコンであることを特徴とする請求項２に記載のパッチ。   The patch according to claim 2, wherein the hydrophobic substance is a hydrophobic plastic or a hydrophobically treated silicon. 前記疎水性物質はポリエチレン、ポリプロピレン、ポリカーボネート、ポリスチレン、ポリメチルメタクリレート、ポリジメチルシロキサン、ポリ塩化ビフェニル、疎水性に処理されたシリコンウェーハ、疎水性に処理されたＩＴＯガラス、およびこれらの組合わせからなる群から選択されることを特徴とする請求項２に記載のパッチ。   The hydrophobic material includes polyethylene, polypropylene, polycarbonate, polystyrene, polymethyl methacrylate, polydimethylsiloxane, polychlorinated biphenyl, a hydrophobically treated silicon wafer, a hydrophobically treated ITO glass, and combinations thereof. The patch according to claim 2, wherein the patch is selected from the group. 前記接着部支持体は、ポリエチレンテレフタレート、ポリエチレン及びポリエステルからなる群から選択されることを特徴とする請求項１に記載のパッチ。   The patch according to claim 1, wherein the adhesive support is selected from the group consisting of polyethylene terephthalate, polyethylene, and polyester. 前記第１接着物質の接着力は、４９０Ｎ／ｍ以上であることを特徴とする請求項１に記載のパッチ。 The patch according to claim 1, wherein an adhesive force of the first adhesive substance is 490 N / m or more. 前記第２接着物質の接着力は、１３０Ｎ／ｍないし２７２Ｎ／ｍであることを特徴とする請求項１に記載のパッチ。 The patch according to claim 1, wherein the adhesive strength of the second adhesive material is 130 N / m to 272 N / m . 前記チャンバ形成部は流線型であることを特徴とする請求項１に記載のパッチ。 Patch according to claim 1, wherein the chamber forming portion is streamlined. 生体分子が固定化されたマイクロアレイ基板上に請求項１ないし８のうちいずれか１項に記載のパッチを付着させて反応チャンバを形成する段階と、
前記反応チャンバに反応液を注入して反応を行う段階と、を含む該パッチの使用方法。 Forming a reaction chamber by attaching the patch according to any one of claims 1 to 8 on a microarray substrate on which a biomolecule is immobilized;
Injecting a reaction solution into the reaction chamber and performing a reaction. 前記マイクロアレイは、蛋白質用マイクロアレイまたはポリヌクレオチドアレイであることを特徴とする請求項９に記載の方法。   The method according to claim 9, wherein the microarray is a protein microarray or a polynucleotide array.
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